The goal of this project is to develop therapeutic radiopharmaceuticals based on targeting the decay of Auger electron emitting radioisotopes to specific sequences in DNA (genes) using triplex forming oligonucleotides (TFOs) as delivery vehicles.In in vitro studies we have demonstrated that TFOs are able to deliver Auger electron emitters to specific targets in cellular DNA in order to inactivate genes and/or kill the cells containing the target sequences. Decay of I=125 in TFOs results in strand breaks in both strands of the target DNA with an efficiency from 0.4'0.8 break/decay. Higher efficiency can be achieved with radionuclide multiple labeling. Breaks are confined to the triplex target sequence, and 90 percent of the sequence specific breaks are located within 10 bp around the decay site. Specificity of TFOs was shown to be high enough to specifically break genomic DNA in a target located in a single copy gene. A liposome delivery system has been developed to effectively deliver radiolabeled TFOs into the cell nucleus. Radiotoxicity of TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated IUdR=125.We have developed a rapid procedure for incorporation of the short life Auger electron emitters I=125 and In=111 into ODNs and demonstrated that decay of these clinically relevant radioisotopes produces DNA breaks with the yield comparable with that of I=125.A new generation of chemically modified TFOs with increased in vivo stability permitting one step labeling with Auger electron emitters is being developed. We have also shown that the fine structure of DNA damage by decay of Auger electron emitter depends on local DNA conformation and, therefore, by analyzing the DNA damage one can obtain information on the structure of DNA in nucleoprotein complexes both in vitro and in vivo. Based on this principle a new method of radioprobing of DNA-protein complexes has been demonstrated in several model systems.In addition, studies have been initiated to investigate the mechanisms of Auger electron-induced DNA strand break repair in human cells. We have developed efficient methods of producing and isolating specific forms (form I and form II) of damaged shuttle vector plasmid DNA, using both oxidative agents and TFO-bound Auger emitting radionuclides as damaging agents. A liposome delivery system has been developed for efficient delivery of damaged DNA into human cells in order to evaluate the in vivo repairability and mutagenicity of site-specific DNA double strand breaks induced by I=125-labeled TFOs. Methods have been developed to recover Auger emitter damaged DNA following intracellular repair in human cells, and evaluate the mutational spectrum and the overall mutagenicity of the Auger emitter induced DNA double strand breaks.In vitro double strand break (DSB) repair assays have been developed to permit isolation of human proteins involved in DSB repair from cell free extracts. Products of reactions using proteins identified by this assay will be examined at the molecular level and compared with products of DNA repaired in vivo role in overall DSB repair.The aim of these studies is to identify the human repair pathways involved in Auger emitter induced DSB repair, assess the consequences of repairing these lesions, and to examine methods by which these repair processes may be manipulated to augment the radiotherapeutic effects of Auger electron emitting TFOs.